Catabolic Phenotype Induction of NP and AF Cells in 3D Culture

Bermudez-Lekerika, P; Croft, AS; Crump, KB; Wuertz-Kosak, K; Le Maitre, CL; Gantenbein, B (2022). Catabolic Phenotype Induction of NP and AF Cells in 3D Culture. In: eCM20: Cartilage and Disc Repair and Regeneration. Davos, Switzerland. 15-18 June.

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Intervertebral disc (IVD) degeneration is considered a key contributor for low back pain, which is the leading cause of disability worldwide1 . In the last decades, a wide range of cytokines have been applied to IVD cells in-vitro to investigate the degenerative cascade2,3 . However, a generic stimulation model defining dose and duration is lacking. Hence, we aimed to explore the catabolic response of bovine IVD cells after TNF-ߙ or IL-1β stimulation at different concentrations and timepoints in-vitro.

METHODS: Primary nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from bovine tails and expanded for two weeks. Afterwards, IVD cells were encapsulated in 1.2% alginate beads (4 x 10 6 cells/ml) and cultured for two weeks for phenotype recovery. Then, alginate beads were stimulated with 0.1, 1 and 10 ng/ml TNF-ߙ or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation. A nonparametric distribution was assumed and Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed.

RESULTS: A dose-dependent upregulation of catabolic markers was observed in NP and AF cells after one day of TNF-ߙ or IL-1β stimulation. Particularly, 10 ng/ml TNF-ߙ stimulation revealed a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells compared to the control group after one day of stimulation. Although the significance was only observed in AF cells, MMP3 upregulation in NP cells showed a strong trend (p=0.0643) (Fig. 1). Nevertheless, the dose-dependent upregulation tendency was less pronounced or even lost after seven days of treatment. In addition, no significant difference between treatments was observed in COL2,

COL1 and ACAN expression. Noteworthy, the cell viability was not reduced after seven days in both NP and AF cells regardless of the treatment.

Fig. 1: Relative gene expression of MMP13 ADAMTS4 and MMP3 in AF and NP cells after one day of stimulation. Shown are means + SD,

N = 2–4, p-value: * <0.05.

DISCUSSION & CONCLUSIONS: We demonstrate a dose dependent upregulation of catabolic markers in NP and AF cells under TNF-ߙ or IL-1β stimulation. Moreover, the significant upregulation of ADAMTS4, MMP3 and MMP13 genes was found in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effect could be counter-regulated due to a possible adaptation of the cells to the catabolic stimuli, suggesting a possible mechanism to face the metabolic shift.

ACKNOWLEDGEMENTS: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735.

REFERENCES: 1 F.J. Lyu, et al (2021) Bone Res 9, 7. 2 K.L.E. Phillips et al (2015) Osteoarthritis and Cartilage 23:1165-77. 3 M.V. Risbud, et al (2014) Nat Rev Rheumatol 10:44-56.

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